Most studies on neuropeptides have been focused on peptides that are synthesized in the secretory pathway and released in a regulated manner when the cell is stimulated. We now refer to these molecules as classical neuropeptides, by analogy to the term classical neurotransmitters that is often used to describe molecules such as acetylcholine and dopamine which are stored in vesicles and secreted on demand, and distinct from non-classical neurotransmitters such as NO and anandamide that are synthesized in a regulated manner in the cytosol and then immediately secreted from the cell. Several lines of evidence point to a new group of signaling molecules, which we refer to as non-classical neuropeptides. These molecules are not produced in the secretory pathway by the conventional peptide processing enzymes, but are instead derived from cytosolic proteins by an unknown mechanism. There have been reports of bioactive peptides that are derived from cytosolic proteins, but the key experiments to establish these as non-classical neuropeptides have not yet been performed. We recently identified a number of hemoglobin-derived peptides in mouse brain, and several of these peptides (N-terminally extended forms of a peptide named hemopressin) were found to be agonists of CB1 cannabinoid receptors. These hemopressin peptides are present in mouse brain at levels comparable to many of the abundant neuropeptides, based on their signal strength in mass spectrometry, and are secreted from brain slices. Furthermore, we have found that hemoglobin mRNA is present in neuronal cultures, and recent reports from two other groups have shown the presence of hemoglobin mRNA and protein in neurons. The critical studies to establish hemopressin as a non-classical neuropeptide will be performed. Aim 1 will examine the enzymatic pathway by which hemopressin peptides are generated and degraded. Aim 2 will examine the mechanism by which hemopressin peptides are secreted from brain cells. Aim 3 will explore the mechanism(s) by which hemopressin peptides are regulated, either at the level of synthesis or secretion. These studies will contribute to a better understanding of the role of non-classical peptides in general, and on the hemopressin peptides in particular, which due to their activity at CB1 cannabinoid are relevant to drugs of abuse. PUBLIC HEALTH RELEVANCE: Neuropeptides function in cell-cell signaling and are produced in the secretory pathway, stored in vesicles, and released in a regulated manner when the cell is stimulated. Several lines of evidence point to a new type of neuropeptide, or non-classical neuropeptide, that is not produced in the secretory pathway by the conventional neuropeptide processing enzymes, but is instead derived from cytosolic proteins. The critical studies to establish if novel cannabinoid receptor agonist peptides (named the hemopressins) represent non-classical neuropeptides are described in this application.